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December 9, 2010

GENETIC ENGINEERING• Genetic engineering or Recombinant DNA technology helps in transferring the genes from one organism toaltogether different organism.• Watson and Crick's discovery of DNA molecular structure and Nathans discovery of Restriction endonucleases laid foundations for genetic engineering.• Recombinant DNA technology uses Vectors to transfer the genes from one organism to another organism.• Genetic engineering is a laboratory technique of gene manipulation, which brings about novel combination of genes.• It helps in obtaining multiple copies of desired gene, which is known as Gene cloning. Gene cloning is carriedout in a computerised machine known as Thermocycler that uses the method called Polymerase Chain Reaction.Method• There are 4 stages in the method of genetic engineering known as1. Isolation of Gene.2. Insertion of gene into suitable vector.3. Introduction of recombinant vector into suitable host. 4. Selection of transformed host cells.1. Isolation of Gene• Plant cells are subjected to cell wall degradation by using enzymes to obtain protoplasts.• Unit membranes of the cell protoplasts are dissolved by using detergents to obtain protoplasm mass. • Protoplasm is treated with phenols and nucleases and later subjected to gradient centrifugation to obtain pureDNA.• DNA is subjected to restriction endonucleases treatment to cut into respective genes or DNA fragments. • Gel Electrophoresis separates these DNA fragments on the basis of their relative length or size.• From a group of genes of the same lengths, the gene with desired sequence is separated by Southern Blottingtechnique. Restriction Endonucleases• These are also known as molecular scissors.• About more than 250 restriction enzymes have been discovered.• These enzymes identify specific nitrogen sequence present between adjacent genes and cut the DNA between two specific adjacent bases of that sequence.• These were discovered by Nathans.• There are two types of Restriction Endonucleases. The first type of enzymes make straight cuts in the DNAmolecule. Hence the DNA fragments formed have blunt or Flush ends. • The second type of enzymes make staggered cuts. Hence the DNA strands are cut at different points of specificsequence known as Palindromic sequence. Hence at one end of the fragment one strand protrudes more than the other and at another strand the other one protrudes more than the earlier one. Such single strandedprotruding ends of the Double stranded DNA fragments are known as Sticky ends or Cohesive ends. Hence theDNA fragments of different organisms generated by the action of same restriction enzyme can be joined with their sticky ends. This property of restriction enzymes has immense value for the formation of recombinant DNA.• In the restriction enzyme Eco RI, E indicates the genus name Escherichia, co indicates the species name coli, R indicates the type of plasmid that has the genes for the enzyme and the roman numerical I indicates the series ofrestriction endonucleases obtained from that organism with that plasmid.• This enzyme recognises the GAATTC sequence on both the strands of DNA and cuts the two strands between G and A.2. Insertion of the isolated gene into a suitable Vector• The desired gene is inserted into suitable vector to produce indefinite number of copies of gene. It is known asgene cloning. The vector acts as a vehicle to carry the gene. • A vector with the characters such as 1. Low molecular weight, 2. Having sites for the action of restrictionenzymes, 3. Ability to replicate inside the host cell and 4. Having genes for antibiotic resistance.• Plasmids, Bacteriophages and Cosmids are some of the commonly used vectors in Genetic engineering. Phage vectors• λ (Lambda) Phage is the most commonly used vector among viral vectors. The advantage of using it is that mostof the phage DNA is not needed for its replication. In place of it the desired gene of donor organism can be inserted.Plasmids• These are extrachromosomal DNA rings present in almost all bacterial species.• They are inheritable and carry few genes. These genes determine variety of functions.• These are very easy to isolate and reintroduce into bacterial cell. • Besides natural vectors, artificially modified plasmids such as pBR 322 (Boliver & Rodriguez) and pUC 19, 101(University of California) are commonly used.• For isolation of plasmid, the bacterial cell is treated with Ethylene Diamine Tetra Acetic acid and lysozyme to dissolve the cell wall. Then the bacterial cell is subjected to centrifugation in Sodium lauryl sulphate solution toseparate the plasmid.Method• The isolated plasmid is cut and opened by subjecting to restriction endonuclease treatment. It is made linear with sticky ends.• The isolated gene is joined to the sticky ends of opened plasmid.• The enzyme DNA ligase forms ester bonds between the ends of DNA strands of plasmid and the isolated geneto form a circular hybrid called Recombinant DNA or Chimeric DNA.3. Introduction of Recombinant Vector into the Host• The recombinant DNA obtained in the above step is brought nearer to a suitable host bacterium in the presenceof dilute CaCl2 solution. It renders the bacterial cell to absorb the DNA and changes into Transformed cell. • The recombinant DNA divides in the host cell and carried to successive generations of bacterial cells. In this wayTransformed bacterial clones are formed.4. Selection of Transformed cells• Selection method depends on the nature of cloned gene. Without using probes:• If the cell with genes for antibiotic resistance has to be selected, the cells first should be incubated for an hour inmedium without antibiotic to make genes for antibiotic resistance expressed. The cells are then placed in a medium containing antibiotic for selection of colonies containing recombinant DNA.• Colony hybridization method is commonly used to identify the cells with desired genes from number differentvarieties of cells or colonies. In this method, labeled probes for a specific gene in the form of single stranded DNA or RNA segments are used. These probes search, and make hydrogen bonds with the desired gene withcomplementary sequence of transformed cells.cDNA• It is complementary DNA synthesized on mRNA in the presence of enzyme reverse transcriptase. It can be used as probe.• It is cloned upon inserting into a vector or directly used to detect the gene of interest.• These are used in the diagnosis of infectious diseases, identification of food contaminants and a variety of microbiological tests.• Forensic tests are conducted by DNA finger printing technology.Transgenic Plants• These are plants into which the genes of unrelated organisms are inserted through genetic engineering methods. • Transgenic plants formation utilises genetic engineering technique and Tissue culture.• Ti (Tumour inducing) plasmid of Agrobacterium tumefaciens is the most commonly used vector in plant geneticengineering. • Though initially genetic engineering was carried out in dicots, now it is extended to even monocots like wheat,maize, rice, oats etc.• Till now about 50 transgenic plants have been produced. But field tests are conducted only in 25 plants. Uses• Transgenic plants show higher crop production because they are resistant to herbicides, insects and viruses.• These plants are suitable for food processing. Delayed ripening and Bruise resistant tomato plants are produced by plant transgenics.• It produced male sterile lines of Brassica napus (Rape seed). Such plants can be directly used as femaleparents in hybridization experiments. It reduces the cost for emasculation for hybrid seed production. • These are used as bioreactors for large-scale production of commercially valuable products, specializedmedicines, chemicals and antibodies. Such usage is known as Molecular farming.• These plants are used for identification of regulatory sequences for many genes.